Micropatterning of biomolecules on surfaces has a number of applications, including the modulation of cell-substrate interactions in biomaterials and tissue engineering and the fabrication of multi-analyte biosensors and genomic arrays. See Blawas, A. S. et al, Biomaterials (1998), 19, 595; Mrksich, M. and Whitesides, G. M., TIBTECH (1995) 13, 228. Microcontact printing (also referred to herein as “μCP”) methods are attractive for micropatterning of biomolecules, because of their simplicity and ease of use. See Kumar, A. .et al., Acc. Chem. Res. 1995, 28, 219; Xia, Y. et al., Angew. Chem. Int. Ed. Engl. (1998), 37, 550. To date, however, methods of microcontact printing have generally been limited to the production of patterns on self assembling monolayers (SAMs), which in turn are bound to gold or silicon surfaces. For example, Whitesides and coworkers have used reactive μCP to pattern biological ligands onto reactive SAMs on gold. J. Lahiri,et al., Langmuir 1999, 15, 2055. Bernard et al. have similarly used μCP to pattern different proteins onto SAMs on gold by physical adsorption. See Bernard, A.; et al., Langmuir 1998, 14, 2225.
U.S. Pat. No. 5,512,131 to Kumar et al. proposes a method of patterning a surface in which an elastomeric stamp with a stamping substrate surface is coated with a self-assembled monolayer-forming species having a functional group selected to bind to a surface. The stamp is then placed against the surface to leave a self-assembled monolayer of the species originally coated onto the stamp. The description of the invention is, however, limited to the use of self-assembling monolayers. While SAMs are commonly used, the limitation of being required to use them is disadvantageous in that SAMs generally bind only to certain materials such as metals (usually gold), silicon dioxide, gallium arsenide, glass, and the like. The patent fails to provide any example of a non-SAM species being used to bind directly to a surface, nor does the patent recite any examples of microcontact printing onto a material other than gold.
While SAMs on gold are generally used for micropatterning, they have limited utility as biomaterials. In contrast, polymers are widely used as biomaterials. (Zdrahala, R. J., J. Biomater. Appl. (1996) 10, 309). Most previous studies on micropatterning on polymers have utilized photolithography. (Mooney, J. F.et al., Proc. Natl. Acad. Sci. (USA) 1996, 93, 12287. Wybourne, M. N. et al., Nanotechnology 1996, 7, 302. Hengsakul, M et al., Bioconj. Chem. 1996, 7, 249. Schwarz, A.et al., Langmuir 1998, 14, 552; Dewez, J.-L.et al., Biomaterials 1998, 19). Alternative methods have also been demonstrated by Ghosh and Crooks, who patterned hyperbranched poly(acrylic acid) on oxidized poly(ethylene) using reactive μCP. (Ghosh, P.; Crooks, R. M. J. Am. Chem. Soc., 1999, 121, 8395).
The micropatterning of biological molecules onto surfaces is an important objective because such patterning enables, for example, control of cell-substrate interactions. (Chen, C. S.; et al., Science 1997, 276, 1425. Mrksich, M.et al., Exp. Cell Res. 1997, 235, 305. Chen, C. S;et al., Whitesides, G. M.et al., Biotechnol. Prog. 1998, 14, 356). In the last decade, biomolecules have been immobilized onto the surface of different polymers in order to modulate their interaction with cells. (Shakesheff, K.et al., J. Biomater. Sci., Polym. Ed. 1998, 9, 507; Cima, L. G., J. Cell. Biochem. 1994, 56, 155; Massia, S. P. et al., J. Biomed. Mater. Res. 1991, 25, 223; Brandley, B. K.; et al., Anal. Biochem. 1988, 172, 270. Massia, S. P. et al., Anal. Biochem. 1990, 187, 292). More recent studies have focused on patterning polymer surfaces with biological ligands. Mooney, J. F.; Hunt, et al., Proc. Natl. Acad. Sci. (USA) 1996, 93, 12287. Wybourne, M. N.et al., Nanotechnology 1996, 7, 302; Hengsakul, M.et al., Bioconj. Chem. 1996, 7, 249; Schwarz, A.; et al., Langmuir 1998, 14, 5526; Dewez, J.-L.et al., P. G. Biomaterials 1998, 19).
Despite the foregoing, current attempts to micropattern biological ligands onto polymer surfaces are severely limited. Most μCP methods are done and indeed are required to be performed on gold or similar metal surfaces. Typically, a SAM-molecule is stamped onto a gold surface to create a patterned SAM layer on the gold surface. See Kumar, A. .et al., supra. In a modification of this basic method, Lahiri et al., supra, have developed a method in which a homogeneous SAM is formed on gold by incubating the gold surface in a solution of the SAM-forming molecules. Next, a stamp is used to transfer a non-SAM reactive molecule to the SAM/gold surface. The reactive molecule reacts with a reactive molecule in the SAM to form a pattern of the reactive molecule on the SAM/gold surface. These methods are limiting because they are restricted to the use of gold or other SAM forming surfaces, and require the use of SAM-forming molecules. These approaches are not applicable to polymer surfaces because SAMs do not generally form on polymers. In yet another alternative approach (Bernard et al., supra), a stamp “inked” with protein is used to stamp a pattern of the protein onto a polymer. A significant limitation of this method is that the protein is not bound to the polymer surface via a stable, covalent linkage or bond. Rather, the protein is attached to the polymer surface by physical adsorption. This approach is limiting because many molecules of interest cannot be stably bound to polymer surfaces by non-specific physical adsorption, and the patterned molecule is easily removed from the polymer surface by water, buffers, biological fluids and the like.
In addition to the above, it has become increasingly important to attempt to control the placement of cells in an organized pattern on a substrate for the development of cellular biosensors, biomaterials, and high-throughput drug screening assays. See e.g., R. Singhvi, G. Stephanopoulos, D. I. C. Wang, Biotechnology and Bioengineering 1994, 43, 764, J. A. Hammarback, S. L. Palm, L. T. Furcht, P. C. Letourneau, J. Neurosci. Res. 1985, 13, 213, and K. E. Healy,B. Lom, P. E. Hockberger, Biotechnology and Bioengineering 1994, 43, 792. A potential problem in spatially directing cellular interactions at a biomaterial surface is the relatively rapid adsorption of a complex layer of proteins within a relatively short period of time of contact with serum in cell culture or upon implantation in vivo. See e.g., T. A. Horbett, J. L. Brash, ACS Sym. Ser. 1987, 343, 1.; J. D. Andrade, V. Hlady, S. I. Jeon, in Hydrophilic polymers: Advances in Chemistry Series, Vol. 248 (Eds: J. E. Glass), ACS, Washington, D.C. 1996, p 51. The adsorbed layer of proteins may potentially physically obscure the micropatterned cell-adhesive ligand, or present a multitude of alternative cellular signals, which has the ability to prevent the formation of cellular patterns, mediated by the micropatterned cell-adhesive ligand.
One proposed approach to address this problem involves the presentation of a biochemical ligand of interest against a protein-resistant, nonfouling surface. A method to prevent nonspecific protein adsorption involves the incorporation of polyethylene glycol (PEG) at the surface. A number of methods have been proposed to incorporate PEG at surfaces, including physisorption (e.g., J. H. Lee, P. Kopeckova, J. Kopecek, J. D. Andrade, Biomaterials, 1990, 11, 455; J. A. Neff, K. D. Caldwell, P. A. Tresco, J. Biomed. Mater. Res. 1998, 40, 511), chemisorption (e.g., K. L. Prime, G. M. Whitesides, Science 1991, 25215, 1164.; K. L. Prime, G. M. Whitesides, J. Am. Chem. Soc. 1993, 115, 10714), chemical grafting (e.g., J. M Harris, in Polyethyleneglycol Chmistry; bioechnicqal and Biomedical Applications: Plenum Press, New York, 1992; K. D. Park, W. G. Kim, H. Jacobs, T. Okano, S. W. Kim, J. Biomed. Mater. Res., 1992, 26, 739.; Y. C. Tseng, K. Park, J. Biomed. Mater Res. 1992, 26, 373.; M. Amiji, K. Park, J. Biomater.Sci., Polym. Ed. 1993, 4, 217), plasma-initiated grafting (e.g., M. S. Sheu, A. S. Hoffman, J. G. A. Terlingen, J. Feijen, Clin. Mater. 1993, 13, 41), and deposition (e.g., G. P. Lopez, B. D. Ratner, C. D. Tidwell, C. L. Haycox, R. J. Rapoza, T. A. Horbett, J. Biomed. Mater. Res. 1992, 26, 415). Most of these methods, however, typically require multiple processing steps that are often optimized for the surface of interest.
Thus, the successful patterning of biological ligands directly onto polymer surfaces using reactive microstamping techniques (e.g., in which reactions between the ligands and the polymer surfaces occur to create a stable covalent bond between the two) has heretofore remained elusive. Accordingly, a need exists for a reliable method of microstamping biological and other ligands directly and covalently onto polymer surfaces that may render the surface biologically nonfouling.